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US6214542B1 - Quantification of indicators of fibrosis - Google Patents

Quantification of indicators of fibrosis Download PDF

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US6214542B1
US6214542B1 US07/963,475 US96347592A US6214542B1 US 6214542 B1 US6214542 B1 US 6214542B1 US 96347592 A US96347592 A US 96347592A US 6214542 B1 US6214542 B1 US 6214542B1
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diagnostic method
tissue
collagens
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molecules
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Gary E. Striker
Liliane J. Striker
Emmanuel Peten
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6851—Quantitative amplification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q2600/00—Oligonucleotides characterized by their use
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Definitions

  • Fibrosing diseases are known to affect many different mammalian organs. Common examples include the kidney (glomerulonephritis), bladder, prostate (benign prostate hypertrophy), lung (emphysema) and liver. But essentially all tissues are affected, in one way or another, by the fibrotic process. This is due mainly to the wide range of different cells types, such as fibroblasts, smooth muscle cells, and even epithelial cells, that are involved in fibrotic disease. The common thread that links all these cells types with fibrotic disease is the synthesis of connective tissue.
  • fibrosis was a terminal and irreversible process consisting of the deposition of intert connective tissue in the scarring process. It is now thought that fibrosis is a dynamic process up to the end stages of disease. In other words, deposition of scar tissue continues until the affected tissue is almost completely replaced by scar tissue. This finding has considerable therapeutic import since even a fairly late diagnosis of the disease may permit an effective implementation of therapy. It remains quite important to make an accurate diagnosis as early as possible, however, in order to minimize the damage to affected tissues. Unfortunately, overt clinical signs of disease, usually marked by the beginning of organ failure, often do not occur until more than one-half of the organ has been scarred.
  • kidney failure One of the devastating manifestations of fibrotic disease is kidney failure.
  • end-stage renal disease in humans is the result of gradual, glomerular scarring known as glomerulosclerosis.
  • the glomerulus is a tuft of capillaries situated at the origin of the vertebrate kidney that is responsible for filtering impurities from the blood, resulting in the formation of urine.
  • a major cause of glomerular scarring is fibrosis resulting from the excessive deposition of extracellular matrix (ECM) components in the glomerular region. Presumably, this deposition results from a deviation in the tightly regulated balance between the synthesis and degradation of the molecules which comprise the ECM. Little is known concerning the molecular basis of this abnormality.
  • ECM extracellular matrix
  • the ECM is a complex network of macromolecules that fills the tissue space between cells. Until recently, it was thought that the ECM provided a relatively inert scaffold on which cells found support. But it is now clear that this structure is intimately involved with the development and function of many cell types.
  • the ECM of vertebrates is made up of two general groups of molecules, glycosaminoglycans (GAG's) and fibrous proteins.
  • GAG's are long polymers of repeating disaccharide units, most of which are covalently linked to protein molecules to form proteoglycans.
  • the fibrous proteins are of two classes, adhesive and structural. The major fibrous protein, collagen, is of the latter type.
  • Type II collagen and type III collagen are each composed of three identical subunits, ⁇ 1(II) and ⁇ 1(III) respectively.
  • Type I collagen is comprised of two ⁇ 1(I) subunits and one ⁇ 2(I) subunit.
  • type IV collagen is comprised of two ⁇ 1(IV) subunits and one ⁇ 2(IV) subunit.
  • the collagen make up of the ECM can differ dramatically and individual collagens may be further modified depending on their location and role.
  • Type IV collagen has been shown to be the major component of the glomerulosclerotic lesion. Morel-Maroger Striker et al., Lab. Invest. 51:181-192 (1984). Type I collagen, which is not normally found in the glomerular ECM, has also been identified by immunofluorescence in the sclerotic lesion. Merritt et al., Lab. Invest. 63:762-769, 1990. In addition, there is evidence linking most other kinds of collagen with the generation of scar tissue. Id.
  • TIMP's tissue inhibitors of metalloproteinases
  • TIMP-1 and TIMP-2 tissue inhibitors of metalloproteinases
  • the overall gross pathology of fibrotic disease is characterized by an increase in tissue rigidity, a concomittant loss of elasticity, and eventual replacement of organ tissue with scar. Such alterations also adversely affect the function of the organ.
  • the specific cellular changes that occur are presently the subject of intense investigation. What can be said is that the deposition of excess ECM, which leads to scar formation, causes substantial changes in the behavior of cells. The reasons for the production and deposition of excess ECM remain largely unknown.
  • Another object of the present invention is to provide a method by which the state of the extracellular matrix environment of a given tissue can be monitored, over the course of time in a single patient, in order to determine the effectiveness of a course of treatment.
  • a diagnostic method comprising the steps of
  • the amount of amplified cDNA molecules is compared with that of a second organism or, alternatively, of the same organism but generated at an earlier time.
  • the obtained tissue is kidney tissue.
  • FIG. 1 is a schematic representation of one aspect of the present invention, including the steps of biopsy, microdissection, reverse transcription, cDNA amplification, and analysis of the amplified products.
  • the present invention permits one to detect even subtle changes in the nature of the extracellular matrix.
  • the mRNA levels corresponding to certain ECM proteins are often seen to increase in sclerotic regions and appear to be independent of cell number. And there is a positive correlation between histologic findings of sclerosis and the amount of certain ECM-related mRNA's.
  • the present invention employs a series of steps through which discrete regions of organ tissue are examined for abnormalities in extracellular matrix synthesis and degradation. These steps include tissue biopsy, microdissection, reverse transcription, and polymerase chain reaction (PCR). The net result of the combination of these steps is two-fold. First, biopsy and microdissection serve to parse out particular tissues for study. And second, reverse transcription and PCR serve to amplify molecules available only in trace amounts in the dissected tissues, thus allowing for their study ex vivo.
  • the present invention may be applied to the study of fibrosis in almost any tissue. Since all tissues contain connective tissue, there will be some cells responsible for the deposition of connective tissue components (e.g., collagens) in each location. Thus, the potential for abnormal scar formation exists and can be studied employing the methods claimed herein.
  • connective tissue components e.g., collagens
  • Biopsy is simply the removal of a small sample of intact tissue for diagnostic, as opposed to therapeutic purposes. In this context, biopsy of tissue allows one to look at tissue that has developed in its normal environment and that was not subject to the effects of manipulations outside the organism. Biopsy also can be performed such that the organism to be studied need not be sacrificed. In fact, most biopsies can be performed percutaneously with a biopsy needle and the assistance of imaging techniques. Thus, discomfort to the patient and damage to the organ are minimized.
  • biopsies can target specific parts of the organism for study, it is often desirable to examine even smaller segments of the tissue, especially when the effects of disease are localized to these areas. This is accomplished by further subjecting the isolated tissue to microdissection.
  • Microdissection is defined as the isolation of small samples of organ tissue in order to facilitate the study of particular subsegments of that organ. The dissected subsegments should provide the basic organizational units of such tissues and remain essentially intact following microdissection in order to allow results to be correlated with subsegment function. In each tissue there are usually a number of distinct subsegments with differing functions. By examining a discrete subsegment, one can focus attention on a particular function or set of functions associated therewith.
  • Microdissection also provides a further advantage in that the use of such small samples minimizes the negative effects on the subject.
  • RNA is subject to cleavage by highly active enzymes called RNAses. These enzymes are ubiquitous, nearly impossible to remove from samples, and highly resistant to inactivation.
  • RNAses highly active enzymes
  • RNA's cannot be cloned and amplified in the same manner as DNA because they are found in single-stranded form.
  • mRNA levels like those of type IV collagen, are so low as to not be detectable by in situ hybridization.
  • RNA's can be converted into complementary DNA (cDNA) homologs which can be manipulated with greater ease.
  • cDNA's may also be subjected to PCR, described more fully below, allowing for their rapid amplification. Moriyama et al. (1990).
  • Reverse transcriptase requires both a template and a free nucleic acid 3′-terminus to begin transcription.
  • mRNA provides the template. While any nucleotide sequence complementary to the mRNA may provide the 3′-terminus, many times the coding sequence of the RNA is not known.
  • the poly-A tail (a string of deoxyadenosine residues found on most mRNA's which confers stability on the molecule) provides a ubiquitous sequence which can be used to hybridize with a poly-T (deoxythymidine) oligonucleotide. This poly-T molecule “primes” the reverse transcription and, hence, is referred to as a primer.
  • random primers may be used to provide the necessary 3′-terminus.
  • reverse transcriptase can use the remaining hairpin-loop structure at the 3′-end of the single-stranded DNA as a primer for the complementary DNA strand.
  • S1 nuclease cleaves the hairpin and creates a normal double-stranded DNA molecule.
  • Polymerase chain reaction has revolutionized molecular biology in the relatively short time it has been available as an experimental tool.
  • This process allows minute amounts of nucleic acids to be amplified by more than one million-fold, so long as some part of the sequence to be amplified, usually 15 to 20 nucleotides in length, is known. Peten et al., Am. J. Physiol., in press (1992). Yet this process can be performed in minutes, unlike the laborious efforts involved in cloning of DNA. It is most useful where, as is the case with collagen message cDNA's, the amount of target nucleic acid is particularly low.
  • a general strategy in accordance with the present invention, is to create oligonucleotide sequences which are complementary to a double-stranded nucleic acid to be amplified.
  • One primer is generated which hybridizes to the “plus” or “coding” strand of nucleic acid.
  • a second primer is made which hybridizes to the “minus” or “non-coding” strand.
  • the second primer must also be located “3′” to or “downstream” of the first. After repeated rounds of hybridization, extension of the oligonucleotides with a modified DNA polymerase, denaturation, and rehybridization, the sequences located between the two primers are amplified.
  • the present invention is conducted in a manner so as to exploit further the power of PCR technology.
  • Use of a competing DNA fragment in PCR provides a reliable and reproducible method for quantitation of the amount of target DNA in the sample.
  • the competing fragment is a mutated version of the DNA to be amplified containing an engineered restriction endonuclease site. Digestion of the PCR products with the appropriate enzyme allows the total amplified DNA to be characterized as having originated either from the target or competing mutant DNA segments. Comparison of the relative amounts of these two populations allows for a determination of the starting amount of the target sequence.
  • the diagnosis achieved by the present invention will provide information on the progression of fibrotic disease not formerly within the reach of the physician.
  • the physician may tailor treatment for the specific stage of the illness. For example, it may turn out that treatments effective at limiting end-stage fibrosis are ineffective or even harmful to tissue that is only mildy affected.
  • therapy directed at reversing the early stages of fibrosis may not be helpful in treating a severly fibrotic tissue.
  • the present invention will provide more specific information on the make-up of the fibrotic lesion. This information will generally fall into two categories. While only two collagens (Types I and IV) have been demonstrated as components of the fibrotic lesion, other collagens are known to be important in normal scar formation. Therefore, it is important to determine which collagens, or other as of yet undefined molecules, are present in a particular lesion. It is also possible that different collagens may be involved at different stages or different kinds of fibrotic disease. Thus, knowing the presence or absence of certain collagens and the relative amount of these molecules in the fibrotic lesion may prove important. This information is also likely to have a bearing on the nature and duration of therapy.
  • the ability to test a particular patient over an extended period of time permits the physician to obtain a dynamic view of the disease process.
  • the physician instituting therapy asks 1) whether the patient is responding, 2) whether the dosage is appropriate, and 3) when may the therapy be terminated.
  • the present invention provides the physician, for the first time, a way of answering these questions. Therefore, not only does the physician gain the opportunity for early intervention, but he or she may more effectively apply treatment once the disease state has been uncovered.
  • Kidney tissue was obtained from 5-24 wk B6xSJL F1 male and female mice weighing from 11-30 gm.
  • Kidney tissue was obtained from nine patients undergoing nephrectomy for renal cancer and from one patient undergoing open biopsy for nephrotic syndrome and severe renal failure of unknown duration.
  • mice After anesthesia with Avertin (12 ⁇ l IP 1:80 solution/g of body weight), the mice were killed by decapitation. The dissection was carried out as described by Moriyama et al. (1990), using a microdissecting microscope (Wild, Heerbrugg, Switzerland).
  • the kidney was flushed with 3 ml of dissection solution A (4° C.; 135 mM NaCl; 1 mM Na 2 HPO 4 ; 1.2 mM Na 2 SO 4 ; 1.2 mM MgSO 4 ; 5 Mm KCl; 2 mM CaCl 2 ; 5.5 mM glucose; and 5 mM Hepes, pH7.4) and then with 3 ml of the same solution containing 1 mg/ml collagenase (Type I, 300 units/mg, Sigma Chemicals, St Louis, Mo.) and 1 mg/ml bovine serum albumin (molecular biology grade, Boehringer Mannheim, Indianapolis, Ind.). A small, superficial cortical fragment was incubated at 37° C. ⁇ 35 min in the collagenase solution in the presence of O 2 .
  • dissection solution A 4° C.; 135 mM NaCl; 1 mM Na 2 HPO 4 ; 1.2 mM Na 2 SO 4 ; 1.2 m
  • VRC vanadyl ribonucleoside complex
  • the cortical fragment was transferred to a microdissecting dish cooled to 4° C. and glomeruli were separated from tubules and afferent and efferent arterioles in buffer containing 10 mM VRC.
  • the cortical fragments were placed at 4° C. in a particulate RNAse inhibitor solution of VRC. The fragments were transferred to a microdissecting dish cooled at 4° C. and glomeruli were separated from tubules and arterioles in the same RNase inhibitor solution.
  • Murine The isolated glomeruli were washed free of tissue debris and VRC in a second dish containing the original dissection solution at 4° C., and transferred to a siliconized PCR tube containing 10 ⁇ l of the dissection solution with 5 mM dithiothreitol (DTT) and 1.2 unit/ ⁇ l of human placental RNase inhibitor (Boehringer Mannheim,).
  • DTT dithiothreitol
  • human placental RNase inhibitor Boehringer Mannheim, The tubes, containing single or pooled glomeruli, were briefly centrifuged at 15,000 RPM at room temperature to pellet the glomeruli (FIG. 1 ).
  • RNAzol method (Cinna/Biotecx, Laboratories International Inc., Friendswood, Tex.) of RNA extraction was used with tRNA added as a carrier. Immediately before reverse-transcription the RNase inhibitor solution was removed from the PCR tubes and replaced with 9 ml of 2% Triton X-100 containing 1.2 unit/ ⁇ l of RNase inhibitor and 5 mM DTT. A cDNA synthesis kit (Boehringher Mannheim) was used. Oligo dT (15 mers) or random primers were used to prime the reverse-transcription for subsequent mRNA amplification. The reaction mixture was incubated for 60 min at 42° C., then stopped by cooling at 4° C. ⁇ 10 min, and heat-treated at 90° C. for 7 min to inactivate the reverse transcriptase enzyme. Samples were stored at ⁇ 70° C. or ⁇ 20° C. for subsequent manipulations.
  • Murine Since the size of glomeruli varied, pools of glomeruli were prepared. Five separate pools of 7 glomeruli per mouse were individually reverse-transcribed, and the cDNA's from 35 glomeruli pooled in a single tube. The cDNA solution was shown to be homogeneous in preliminary experiments, therefore subsequent manipulations were performed using fractions of cDNAs prepared from pooled glomeruli.
  • Murine Primers for reverse transcription, 22-24 mers, were synthesized on a PCRMate (Applied Biosystems, Foster City, Calif.), purified by affinity column elution, and kept at ⁇ 20° C. in water.
  • the mouse ⁇ 2IV collagen primers were designed in the 3′-untranslated region (UTR) described by Saus et al. in J. Biol. Chem. 264:6318-6324 (1989)).
  • the sense primer corresponded to bp 5562-5584 (SEQ ID NO:1:5′ACT CAT TCC AAC CGT CTG TCA GC 3′) and the antisense primer to bp 6100-6123 (SEQ ID NO:2:5′GCA AAT CAT TGA CAG TGG CGT CTA 3′).
  • the mouse ⁇ 1IV collagen primers were also designed in the 3′-UTR. Muthukumaran et al., J. Biol. Chem. 264:6310-6317 (1989).
  • the sense primer encompassed bp 5809-5830 (SEQ ID NO:3:5′TAG GTG TCA GCA ATT AGG CAG G 3′) and the antisense bp 6271-6292 (SEQ ID NO:4:5′CGG ACC ACT ATG CTT GAA GTG A 3′).
  • the sizes of the corresponding amplified products were 562 bp for the ⁇ 2IV collagen primers and 484 bp for ⁇ 1IV collagen primers, as predicted from the corresponding cDNA sequences.
  • Two additional antisense oligonucleotides were synthesized to serve as amplification product-specific probes for ⁇ 2IV collagen spanning bp 5781-5804 (SEQ ID NO:5:5′CCT GCA GTC TTC CTA AAA TGA GGC 3′) and for ⁇ 1IV collagen spanning bp 6172-6193 (SEQ ID NO:6:5′GCA TTT CAC ACC TGA GCA CAC A 3′). Both oligonucleotides localized to a sequence internal to the amplification primers.
  • Sense and antisense primers designed using the previously published cDNA sequence for human TIMP-1 of Carmichael et al., (1986), were synthesized on a PCR-Mate (Applied Biosystems, Foster City, Calif.). A primer pair was chosen to yield an expected product of 442 base pairs.
  • the sense primer sequence was 5′AAT TCC GAC CTC GTC ATC AGG 3′(SEQ ID NO:7) (bp 181-198), and the antisense primer sequence was 5′ACT GGA AGC CCT TTT CAG AGC 3′(SEQ ID NO:8).
  • the ⁇ 2IV collagen primers were designed in the coding region of the NC1 domain in which nucleotide sequences allowed specific amplification of the ⁇ 2 chain, but no other type IV collagen ⁇ chain.
  • PCR was performed using the GeneAmp DNA Amplification kit (Perkin Elmer Cetus, Norwalk, Conn.). The PCR reaction was conducted as described by Moriyama, et al. (1990), but in a final volume of 50 ⁇ l. The final magnesium concentration was 1.5 mM for ⁇ 1IV and 2.0 mM for ⁇ 2IV collagen mRNA amplification. An initial reaction was conducted to determine the range and relative amounts of mutant and test cDNAs to be added to each tube and the number of PCR cycles required.
  • PCR assays were set up as follows: (1) a master mix containing that amount of glomerular cDNA representing ⁇ fraction (1/10) ⁇ th of a glomerulus to be tested and all the PCR kit reagents was prepared and distributed in 45 microliters aliquots into each of 6 PCR tubes (1 additional tube contained all the reagents but no cDNA, as a contamination control); (2) 5 ⁇ l of mutant cDNA template in decreasing concentrations were added to the 6 PCR tubes, usually spanning a 1 to 10 attomole range; (3) the PCR cycles were completed, usually in the range of 36.
  • the thermal cycler (Perkin Elmer Cetus) was programmed so that the first incubation was performed at 94° C. ⁇ 3 min, followed by 3236 cycles consisting of the following sequential steps:94° C. ⁇ 1 min (denaturation), 60° C. ⁇ 1 min (annealing), and 72° C. ⁇ 3 min (extension). The final incubation was performed at 72° C. ⁇ 7 min. Control tubes omitted the reverse transcriptase enzyme to confirm that relevant genomic DNA was not amplified.
  • Murine A quantitative method was established by developing mutated cDNA templates of ⁇ 1IV and ⁇ 2IV collagen cDNAs which would compete with test cDNA on an equimolar basis. Gilliland et al., Proc. Nat'l Acad. Sci. USA 87:2725-2729 (1990). Briefly, templates for ⁇ 1IV and ⁇ 2IV collagen cDNAs were synthesized using PCR site-specific DNA mutagenesis. Ho et al., Gene 77:51-59 (1989). The mutated template for ⁇ 1IV collagen contained a point mutation at bp 6028 (DATP to dCTP) which resulted in a new, unique BclI restriction site.
  • bp 6028 DATP to dCTP
  • the concentration of the purified mutants was determined by comparison with standards (GelMarker I, Research Genetics, Huntsville, Ala.), by densitometric scanning of agarose gels. Serial dilution standards of mutant templates (1000 to 0.0001 attomoles/ ⁇ l) were stored at ⁇ 20° C.
  • the quantitative PCR method for human ⁇ 2IV collagen was facilitated by the use of a mutant cDNA template.
  • the mutant was synthesized by PCR with a 69 bp deletion in the middle of the ⁇ 2IV collagen cDNA molecule and purified by phenol/chloroform extraction from a low melting point agarose gel. The concentration of the purified mutants was determined by comparison with standards (GelMarker I, Research Genetics, Huntsville, Ala.), by densitometric scanning of agarose gels. Serial dilution standards of mutant templates (1000 to 0.0001 attomoles/ ⁇ l) were stored at ⁇ 20° C.
  • the densitometric values of the test and the mutant band(s) were calculated and their ratio for each reaction tube was plotted as a function of the amount of mutant template added. A straight line was drawn by linear regression analysis. The quantity of cDNA in the test sample was calculated to be that amount at which the mutant/test band density ratio was equal to 1. Gilliland et al. (1990). Competitive PCR assays were performed in duplicate or triplicate.
  • Amplified mutant DNA was distinguished from wild-type cDNA on the basis of differring molecular weights. Where necessary, the amplified cDNA's were treated with the appropriate restriction endonuclease to visualize the differences engineered into the sequences. Upon electrophoresis, mutant DNA's migrated at different rates than the wild-type cDNA's, allowing for determination of the relative amounts of the two DNA populations. Knowledge of the starting concentration of the mutant DNA population allowed for a determination of the starting amount of the wild-type cDNA population.
  • DNA was transferred to a Nylon membrane (Schleicher and Schuell, Keen, N. H.) with a vacuum blotter (Hoef fer Scientific Instruments, San Francisco, Calif.) and UV-crosslinked (Stratalinker, Stratagene, La Jolla, Calif.). Synthetic oligonucleotide probes described above were end-labeled with 32 P as described previously. Moriyama, et al., 1990). The amplified products were identified as specific for mouse ⁇ 1IV or ⁇ 2IV collagen mRNA and ⁇ 1IV collagen.
  • Murine Analysis of variance (ANOVA) was used with Bonferroni's post test p corrections for comparisons involving more than two groups, whereas unpaired Student't test was otherwise performed. A p value ⁇ 0.05 was considered significant. All data are expressed as mean ⁇ standard error of the mean (SEM), unless otherwise specified.

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US20060182716A1 (en) * 2004-08-09 2006-08-17 Jin Hong Synthetic hyperglycosylated, protease-resistant polypeptide variants, oral formulations and methods of using the same
US20060204473A1 (en) * 2004-08-09 2006-09-14 Blatt Lawrence M Synthetic hyperglycosylated, and hyperglycosylated protease-resistant polypeptide variants, oral formulations and methods of using the same
US20070117841A1 (en) * 2003-10-24 2007-05-24 Ozes Osman N Use of pirfenidone in therapeutic regimens
US20080260820A1 (en) * 2007-04-19 2008-10-23 Gilles Borrelly Oral dosage formulations of protease-resistant polypeptides
EP2390262A1 (fr) 2003-05-16 2011-11-30 Intermune, Inc. Ligands de récepteur de chimiokine synthétique et leurs procédés d'utilisation
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US5928870A (en) * 1997-06-16 1999-07-27 Exact Laboratories, Inc. Methods for the detection of loss of heterozygosity
US6100029A (en) * 1996-08-14 2000-08-08 Exact Laboratories, Inc. Methods for the detection of chromosomal aberrations
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